Project Summary Chronic pain is a leading cause of global disability, with back and neck pain, migraine and arthritis impacting quality-of-life of millions of individuals worldwide. Despite the huge unmet medical need, existing treatments are often ineffective or are associated with unwanted consequences. Chronic pain is maintained, in part, by persistent changes in sensory neurons. These changes include a pathological increase in peptides derived from the neurosecretory protein VGF (non-acronymic). We have shown that the C-terminal peptide, TLQP-62, is contributes to spinal neuroplasticity and that TLQP-62 immunoneutralization attenuates established mechanical hypersensitivity in the spared nerve injury (SNI) model. The central hypothesis of this project is that spinal TLQP-62 signaling can be targeted for development of new chronic pain treatments through immunoneutralization and/or receptor inhibition. We propose to pursue discovery and validation of TLQP-62- based therapeutic interventions along two parallel lines: 1) identification of TLQP-62 receptor(s) and 2) validation of anti-TLQP-62 antibodies as a potential biological treatment. Pursuing these objectives in parallel maximizes the likelihood of identifying a promising therapeutic intervention in this high-risk project. Currently, there is no information on putative TLQP-62 receptor(s), but our preliminary data show that TLQP-62 activates Ca2+ transients in primary afferent neurons. This suggests that sensory neurons express TLQP-62 receptors, creating an opportunity for a focused receptor identification strategy. We expect this strategy will lead to the discovery and initial validation of a novel receptor target for chronic pain treatment. However, if the effects of TLQP-62 are mediated by multiple receptor types, peptide immunoneutralization may be a more effective treatment than the targeting of one receptor. In Aim 1, we will validate TLQP-62 immunoneutralization as a strategy for chronic pain by a) independently reproducing the efficacy in the SNI model, and b) extending the validation to the SPARC-null mouse model of low back pain associated with intervertebral disc degeneration. In Aim 2, we will identify putative receptor(s) by cross-linking a modified peptide to binding partners, characterization of complexes by Western blot, and identification by mass spectrometry. The top candidate receptor will be validated as a potential target for chronic pain using RNAscope to determine its tissue distribution, and shRNA-mediated knockdown to define its function in TLQP-62-induced Ca2+ signaling and nerve injury-induced hypersensitivity. This discovery and validation project will advance the goal of identification and validation of non-opioid therapies for chronic pain treatment ? the TLQP-62 and its putative receptor(s).